Probing the substrate specificity of Trypanosoma bruceiGlcNAc-PI de-N-acetylase with synthetic substrate analogues

• Published in: Organic & biomolecular chemistry 2014-02, Vol.12 (12), p.1919-1934
• Main Authors: Capes, Amy S, Crossman, Arthur, Urbaniak, Michael D, Gilbert, Sophie H, Ferguson, Michael AJ, Gilbert, Ian H
• Format: Article
• Language: English
• Subjects: Trypanosoma brucei
• ISSN: 1477-0520; 1477-0539
• DOI: 10.1039/c3ob42164c

A series of synthetic analogues of 1-d-(2-amino-2-deoxy- alpha -d-glucopyranosyl)-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glyce rol 3-phosphate), consisting of 7 variants of either the d-myo-inositol, d-GlcpN or the phospholipid components, were prepared and tested as substrates and inhibitors of GlcNAc-PI de-N-acetylase, a genetically validated drug target enzyme responsible for the second step in the glycosylphosphatidylinositol (GPI) biosynthetic pathway of Trypanosoma brucei. The d-myo-inositol in the physiological substrate was successfully replaced by cyclohexanediol and is still a substrate for T. bruceiGlcNAc-PI de-N-acetylase. However, this compound became sensitive to the stereochemistry of the glycoside linkage (the beta -anomer was neither substrate or inhibitor) and the structure of the lipid moiety (the hexadecyl derivatives were inhibitors). Chemistry was successfully developed to replace the phosphate with a sulphonamide, but the compound was neither a substrate or an inhibitor, confirming the importance of the phosphate for molecular recognition. We also replaced the glucosamine by an acyclic analogue, but this also was inactive, both as a substrate and inhibitor. These findings add significantly to our understanding of substrate and inhibitor binding to the GlcNAc-PI de-N-acetylase enzyme and will have a bearing on the design of future inhibitors.